Project Summary Extracellular vesicles (EVs) are membrane-wrapped structures containing proteins, RNAs, lipids, and metabolites that are released from most if not all cell types to mediate intercellular communication. Roles for EVs in physiological processes as well as pathological conditions including neurodegenerative diseases and cancer have been established. Given the presence of EVs in diverse body fluids, there is also great interest in using these vesicles as biomarkers for disease detection and engineering EVs for therapeutics. Investigation of the release of EVs containing fluorescently-tagged cargo from identified cells in the model system C. elegans can provide insight into unresolved questions concerning conserved mechanisms of EV biogenesis and cargo selection in vivo. We discovered that the calcium homeostasis modulator ion channel CLHM-1 is cargo in EVs released from cilia of male-specific sensory neurons. Remarkably, when we coexpressed tdTomato- tagged CLHM-1 with GFP-tagged PKD-2, a known EV cargo protein expressed in the same neurons, we rarely observed colocalization of the fluorescent proteins in vesicles, suggesting that CLHM-1 and PKD-2 are in distinct EV subpopulations. We have found that the PKD-2 and CLHM-1 containing EVs do not utilize the same biogenesis and release mechanisms, are discharged in different quantities, and do not have the same physiological function. Our overarching goal is to draw upon the strengths of our genetic system and cutting edge imaging and mass spectrometry approaches to define mechanisms underlying formation of EV subpopulations and the physiological significance of EV heterogeneity. Our proposed research will utilize our unique transgenic animals that express fluorescently tagged EV cargoes at endogenous levels. Advanced imaging techniques including confocal microscopy with Airyscan detection and immunogold labeling for transmission electron microscopy will enable us to characterize the size, morphology, and ciliary release site(s) of EVs as well as the impact of lateral lipid asymmetry in the ciliary membrane on cargo sorting. Through a candidate approach, we will define the role of flippases, floppases and scramblases, which control transbilayer lipid asymmetry, in the biogenesis of the EV subsets. We will then explore how cellular stress conditions that disrupt plasma membrane phospholipid homeostasis impact EV cargo sorting and release. To identify other cargoes in the CLHM-1 EV subset, we will perform mass spectrometry on GFP-tagged CLHM-1 vesicles isolated by flow cytometry. Finally, we will identify the hermaphrodite-derived stimulus that induces an increase in formation of CLHM-1 containing EVs from male ciliated neurons as well as the importance of EV release for animal communication and ciliary function. This work will lead to an understanding of how an individual cell generates heterogeneous EV populations with different physiological functions, impacting broadly on our comprehension of basic biogenesis and cargo sorting mechanisms utilized in vivo.